Could Public Restrooms Be an Environment for Bacterial

Resistomes?
Hermine V. Mkrtchyan1, Charlotte A. Russell2, Nan Wang1,3, Ronald R. Cutler1*
1 Pre-Clinical Drug Discovery Group, School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom, 2 School of Biological and
Chemical Sciences, Queen Mary University of London, London, United Kingdom, 3 School of Life Science and Technology, University of Electronic Science and Technology,
Chengdu, China

Abstract
Antibiotic resistance in bacteria remains a major problem and environments that help to maintain such resistance, represent
a significant problem to infection control in the community. Restrooms have always been regarded as potential sources of
infectious diseases and we suggest they have the potential to sustain bacterial resistomes. Recent studies have
demonstrated the wide range of different bacterial phyla that can be found in non-healthcare restrooms. In our study we
focused on the Staphylococci. These species are often skin contaminants on man and have been reported as common
restroom isolates in recent molecular studies. We collected samples from 18 toilets sited in 4 different public buildings.
Using MALDI-TOF-MS and other techniques, we identified a wide range of antibiotic resistant Staphylococci and other
bacteria from our samples. We identified 19 different Staphylococcal species within our isolates and 37.8% of the isolates
were drug resistant. We also identified different Staphylococcal species with the same antibiograms inhabiting the same
restrooms. Bacterial resistomes are communities of bacteria often localised in specific areas and within these
environments drug resistance determinants may be freely transferred. Our study shows that non-healthcare restrooms are
a source of antibiotic resistant bacteria where a collection of antibiotic resistance genes in pathogenic and non-pathogenic
bacteria could form a resistome containing a nexus of genetic diversity
Citation: Mkrtchyan HV, Russell CA, Wang N, Cutler RR (2013) Could Public Restrooms Be an Environment for Bacterial Resistomes? PLoS ONE 8(1): e54223.
doi:10.1371/journal.pone.0054223
Editor: Patrick M. Schlievert, University of Iowa Carver College of Medicine, United States of America
Received July 31, 2012; Accepted December 10, 2012; Published January 17, 2013
Copyright: 2013 Mkrtchyan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: r.cutler@qmul.ac.uk

of antibiotic resistance in bacteria and that to restore the efficacy of

antibiotics, this ecological balance had to be adjusted to favor
antibiotic susceptible bacteria rather than antibiotic resistant
bacteria [10,11]. However, despite the huge efforts that have been
made to control and reduce antibiotic use and misuse in man and
in animals [6,12], antibiotic resistance in bacteria continues to
spread and cause morbidity, mortality and increasing costs in the
treatment of infectious diseases [12,13].
In addition to problems with antibiotic misuse however, another
factor which could also be involved with the persistence of drug
resistance in bacteria in the environment, is their ability to form
resistomes, closely associated groups of bacteria able to share
and maintain drug-resistance determinants within suitable environments [1416]. Bacterial resistomes may be associated with
interactions between a wide range of different bacterial species or
even interactions locally between organisms of the same species
[11,16,17].
In our study, the Staphylococcaceae, which are commonly found
associated with restrooms [1], were selected for further study.
However, to fully evaluate the propensity of antimicrobial
resistance in this group of organisms, identification to species
level is required. In the past, full identification of environmental
bacterial species could be difficult and time-consuming as the
majority of high-throughput bacteriological identification systems
were developed for identifying hospital isolates. In our study we
additionally evaluated the use of MALDI-TOF-MS [18,19] for

Introduction
Molecular techniques have recently been used to demonstrate
the wide range of bacterial phyla that can be found in public
restrooms [1]. Previous investigations of restrooms in nonhealthcare environments concentrated primarily on investigating
contamination with bacteria from faecal and/or skin origin [2-5]
and in hospitals the focus was on Staphylococcus aureus or MRSA [6].
The recent study by Flores and co-workers [1] however,
demonstrated the wide diversity of bacterial phyla that can be
present in public restrooms and indicated that these phyla were
usually related to bacteria associated with man. It is therefore not
surprising that organisms associated with the human microbiome [7] should have an impact on the microbial flora in
restrooms [1].
Apart from the presence of human pathogens in restroom
environments, there is also the possibility that restroom environments could harbor antibiotic resistant bacteria, as other studies
looking at non-hospital environments with equally diverse
bacterial populations, have suggested that such populations can
provide effective environments to aid the development, sustainability and spread of antibiotic resistance in bacteria [8]. In
addition to this there is also the suggestion that cells can survive or
persist in such environments even if there are restrictions on
resources [9].
It has been proposed that human societys overuse and abuse of
antibiotics is the main factor in the development and sustainability
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Drug Resistant Bacteria in Nonhealthcare Restrooms

Extracted method. 3-5 colonies of overnight cultures were

suspended in 300 ml distilled water. The suspension was mixed
with 900 ml absolute ethanol and centrifuged for 2min at
130006g. The pellets were re-suspended in 25 ml of 70% formic
acid and then 25 ml pure acetonitrile was added. After mixing
solutions were centrifuged at 130006g for 2 min. 1 ml aliquots of
the supernatant were spotted in duplicate onto MALDI ground
steel targets, dried in air for 5 min at room temperature and each
target spot was overlaid with 1 ml a-cyano-4-hydroxycinnamic
(HCCA) matrix solution.

identifying environmental isolates and compared this method to

selected routine and molecular methods.

Materials and Methods

Sample Collection
Dry sterile cotton swabs (Copan Diagnostics Inc., USA) were
used to collect samples from 18 randomly selected public
restrooms (non-healthcare) in London United Kingdom. Sampling
was carried out in different buildings and over a period of 24
weeks. 21 sites were sampled in each restroom. All specimens were
transferred to the laboratory within 1-3 hrs.of the sample being
taken. In the laboratory, swabs were suspended in 1ml sterile 0.9%
saline, inoculated directly onto Nutrient Agar (Nutrient Agar,
Oxoid, Basingstoke, UK) and plates were incubated aerobically at
37uC for 24-48 h.

Antimicrobial Susceptability Testing

Three culture based methods were used to screen for antibiotic
susceptibilities of Gram-positive cocci and Gram-negative rods.
Mastrings and Microscan Walkaway Plus. Zones of inhibition
were evaluated using Mastring M13 were used for Gram positives
cocci and Mastring M14 for Gram negative rods according to
manufacturers instructions (Mast Diagnostics, Merseyside, UK).
For Gram-positives the minimum inhibitory concentrations (MIC)
were determined using the MicroScan Walkway 96 plus automated system (Siemens Healthcare Diagnostics, CA, USA). The MICs
to oxacillin were additionally evaluated using M.I.C. evaluators,
antimicrobial gradient strips designed for accurate Minimum
Inhibitory Concentration (MIC) values (Oxoid Ltd., Basingstoke,
UK).
For Gram-negative bacteria the minimum inhibitory concentrations (MIC) were also determined using the MicroScan
Walkway 96 plus automated system (Siemens Healthcare Diagnostics, CA, USA).

Identification of the Environmental Isolates

Conventional and biochemical methods. These methods
were used for Gram-positive cocci and Gram-negative rods only.
Gram-positive cocci were provisionally identified by conventional
methods including catalase, coagulase tests and selective media,
(Mannitol Salt Agar, Oxoid Ltd, Basingstoke, UK), Gram negative
rods were provisionally identified using selective media (BRILLIANCETM UTI selective agar, Oxoid Ltd., Basingstoke, UK).
Gram positive cocci were additionally characterised to species
level using the API ID 32 STAPH system (BioMerieux Ltd.,
Marcy lEtoil, France) according manufacturers instructions and
the ProlexTM Staph Xtra Latex Kit was used to distinguish
Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from
other species of staphylococci (Prolab Diagnostics, Neston, South
Wirral, UK).

Detection of PBP2
For resistant staphylococci a rapid latex agglutination assay kit,
the Penicillin-binding protein (PBP2) latex agglutination test was
used (according the manufacturers instructions) to determine
PBP2 (Oxoid Ltd., Basingtoke, UK).

Partial
16S
RNA
gene
sequencing
for
Staphylococci. Genomic DNA of the isolates was prepared

using a commercial kit (Qiagen, Crawley, UK). Staphylococci

were subjected to partial 16S rRNA gene sequencing using
primers described previously [20]. PCR thermal cycling conditions
were 5 min at 94uC, 30 cycles for 30 sec at 94uC, 1 min for 50uC
and 30 sec for 72uC. The 2 log DNA ladder I (New England
Biolab, Hitchin, UK) was used as molecular size markers.
Amplified PCR products were sequenced by Eurofins MWG
GmBH (Ebersberg, Germany) using ABI 37306L DNA analyser.
MALDI-TOF-MS analysis. All isolates were purified and
analysed using Matrix-assisted laser desorption ionization timeflight mass-spectroscopy (Microflex LT, MALDI-TOF-MS, Bruker Daltonics, Coventry, UK) in a positive linear mode (2000 to
20000 m/z range). The resulting spectra for each culture was
analysed by MALDI-Biotyper 2.0 software (Bruker Daltonics,
Coventry, UK). The software evaluates each spectra compared to
a reference spectra in the Bruker Taxonomy Database identifying
the best match from database records. Results were expressed as
scores (QI) from 0 to 3, as recommended by the manufacturer.
Scores QI #1.7 were not considered as reliable identification. A
score of QI$1.7 corresponded to genus identification. Only
scores higher than QI$2 were considered a reliable identification
of species. MALDI-TOF-MS identifications were performed in
duplicate using extracted and direct methods as recommended by
the manufacturer. E. coli DH5 (Bruker Daltonics, Coventry, UK)
was used as a standard for calibration and quality control.
MALDI-TOF-MS analysis Direct method. A single
colony of each overnight culture was transferred onto a MALDITOF-MS ground steel target plate using a disposable loop and
dried for five minutes at room temperature. The HCCA matrix
solution (1 ml) was overlaid onto each target spot.
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PCR Amplification
Genomic DNA of the isolates were prepared using commercial
kits, QIA amp DNA mini kit (Qiagen, Crawley, UK). SCCmec type
was determined by detecting mec and ccr complexes using the
primers as described previously [21]. PCR thermal cycling
conditions were 5 min at 94uC, 30 cycles for 30 sec at 94uC,
1 min for 50uC and 30 sec for 72uC. The 2 log DNA ladder I
(New England Biolab, Hitchin, UK) was used as molecular size
markers.

Results
Samples
Of the 21 sites sampled in each restroom we identified 6 sites
which were the most contaminated. These were the hand dryer
systems, toilet seats, inner door surfaces, taps, soap dispensers and
urinal floors.

MALDI-TOF-MS Analysis
211 of the 256 environmental isolates (82.4%) were identified
using MALDI-TOF-MS (Table 1) however 17.6% of isolates failed
to give a reliable identification. The rates of MALDI-TOF
identification at the species level with a score of QI$2 were 70.7%
(149/211) and at genus level with a score QI$1.7 but QI#2.0
were 29.3% (62/211). Analysis using the direct method resulted in
163 isolates identified, 67.7% of these at species level and 37.3% at
genus level respectively. The alcoholic extraction method significantly improved identification with 185 identified, 83.7% of these
2

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Drug Resistant Bacteria in Nonhealthcare Restrooms

TOF-MS QI score, 2.437. Overall, we identified 103 staphylococcal isolates belonging to 15 species. This included S. aureus,
Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus warneri,
Staphylococcus pasteuri, Staphylococcus simulans, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus cohnii, Staphylococcus capitis,
Staphylococcus pettenkoferi, Staphylococcus lugdunensis, Staphylococcus
arlettae, Staphylococcus equorum, Staphylococcus sciuri. When we compared the MALDI-TOF-MS and PCR sequencing identifications
to those obtained by the API ID 32 STAPH system, the API ID 32
STAPH system misidentified S. pasteuri isolates as S. warneri (one S.
pasteuri was identified as S. hominis) and S. warneri isolates as S.
saprophyticus. One S. haemolyticus was identified as S.hominis and one
S. epidermidis was identified as S. capitis. Only one staphylococcal
isolate (S. cohnii) was misidentified by MALDI-TOF-MS while
PCR sequencing results identified as it S. haemolyticus and by API
test as S. xylosus (Table 3).

Table 1. Summary of Family and Genera of bacteria identified

by MALDI-TOF-MS.

Family

Genus

Staphylococcaceae

Staphylococcus

No of isolates
103

Bacillaceae

Bacillus

37

Micrococcaceae

Micrococcus

30

Enterobacteriaceae

TOTAL (composed of 2)

"

Escherichia

"

Proteus

"

Citrobacter

"

Morganella

Moraxellaceae

Acinetobacter

Corynebacteriaceae

Corynebacterium

Comamonadaceae

Delftia

Sphingobacteriaceae

Sphingobacteria

Campylobacteraceae

Campylobacter

Pseudomonacae

Pseudomonas

Others*

15

TOTAL

211

Antibiotic Susceptibility
Antibiotic resistance was detected both in Gram positive and
Gram negative isolates. Four out of 20 Gram-negative bacteria
species identified were resistant to antibiotics, this included
ampicillin, cephalothin, streptomycin, sulphatriad, tetracycline,
cotrimoxazole (Table 4). In contrast, 39 staphylococcal isolates
(37.8%) were drug resistant, including resistance to non-b-lactam
antibiotics such as fusidic acid, gentamycin, erythromycin and
chloramphenol (Table 5). In addition to these multiply antibiotic
resistant strains, 5 of the staphylococcal strains isolated were
resistant only to oxacillin and penicillin although their oxacillin
MICs were amongst the highest identified, 64 mg/l or greater.
The majority of other strains, although also mecA positive, had
MICs of 4 mg/l or below. Overall MICs for oxacillin varied from
0.25 to 128 mg/l (Table 5).
In some cases there were up to 4 different staphylococcal species
with closely related antibiograms isolated from different sites in the
same restroom. This could be in keeping with the local spread of
drug resistance determinants between related organisms present in
the same restroom. In restroom 16 K for example, 7 antibiotic
resistant staphylococcal strains were isolated from different sites in
this restroom, these were of 4 different species but each with the
same antibiogram. In addition two of these species had multiple
isolates with the same antibiograms, S.epidermidis (2 isolates) and
S.haemolyticus (3 isolates). Overall in restroom 16 K (table 5) the
majority of strains carried 7 of the same class of antibiotic
resistance determinants, (including mec A) common in all 5
isolates.

*Others include genera of Korucia (6); Rothia (2); Arthrobacter (2); Anaerococcus
(3); Rhodococcus (2).
doi:10.1371/journal.pone.0054223.t001

at species level and 16.3% at genus level respectively. This

confirms that best practice for environmental isolates, as
recommended by the manufacturer for clinical isolates, is to use
both direct and extraction methods in combination.
For the Gram negatives tested there was agreement between
conventional methods, 16S DNA and MALDI-TOF-MS analysis
for E. coli and other Gram-negative bacteria such as Proteus mirabilis
and Acinetobacter spp. Both extracted and direct methods were
effective in identifying E. coli and produced high scores of
QI = 2.473 and QI = 2.341 respectively. The alcoholic extraction
method increased identification of Proteus (80%) and Acinetobacter
(85.7%) (Table 2).
In this current study we had a large number of staphylococci in
our samples. We paid particular attention to evaluating the
efficiency of MALDI-TOF to identify environmental staphylococci
and compared the results to conventional identification tests, (API
ID 32 STAPH tests and PCR sequencing). Using the AE method
with MALDI-TOF-MS increased the identification rate for
staphylococci at species level and produced the highest MALDI-

Table 3. Identification of Staphylococci isolates by MALDITOF MS and API ID 32 STAPH system compared with 16S RNA
sequencing.

Table 2. Concordance between conventional and direct and

extracted MALDI-TOF-MS methods for Gram-negative isolates
of three genera.

Genera

Total

AE
D-MALDI- E-MALDI- increase
Conventional TOF
TOF
(%)

E.coli

n/a

Proteus

80

Acinetobacter

85.7

D-MALDI-TOF (direct method); E-MALDI-TOF (extracted method); AE increase

(increase of identification after alcoholic extraction).
doi:10.1371/journal.pone.0054223.t002

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MALDI-TOF MS

API ID 32 STAPH

PCR

S. pasteuri

S. warneri

S. pasteuri

S. pasteuri

S. hominis

S. pasteuri

S. warneri

S. saprophyticus

S. warneri

S. haemolyticus

S. hominis

S. haemolyticus

S. epidermidis

S. capitis

S. epidermidis

S. cohnii

S. xylosus

S. haemolyticus

doi:10.1371/journal.pone.0054223.t003

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Drug Resistant Bacteria in Nonhealthcare Restrooms

identified had not been evaluated in clinical studies [23]. Other

studies using environmental or animal coagulase negative staphylococci (CoNS) have also reported poor identification using API
systems [24,25] [26].
Staphylococci are a major cause of nosocomial and community
acquired infections and they can have a high intrinsic resistance to
antimicrobials [27]. This potential problem could be augmented
by fact that species traditionally regarded as hospital pathogens are
also isolated from non-hospital environments. CoNS in particular,
belong to this group [28]. We sampled 18 restrooms in 4 different
buildings over a period of 24 weeks and found a wide variety of
staphylococcal species, many of which were antibiotic resistant.
The antibiograms of some of these species were closely associated
with the antibiograms found in different species from the same
restrooms on different dates and others with isolates from
restrooms in the same building (table 5). We particularly found
the widespread dissemination of drug resistance in CoNS from
these restroom environments. The numbers of antibiotic resistance
determinants carried by these strains varied from 1 to 15. The
most common resistances were: Penicillin, found in 100% of
isolates, Erythromycin, found in 90%, Amoxillin, 80% and Fusidic
acid, 74%. These resistances were common to isolates in all four
buildings sampled over the four weeks of the study.
Regarding the possibility of the direct transfer of resistance
determinants within restrooms and/or within buildings, there were
11 staphylococcal isolates with the same antibiograms representing
5 different staphylococcal species and these were isolated from 5
different restrooms within the same building (Table 5). The species
(and number of isolates) involved were S.saprophyticus (n = 3),
S.epidermidis (n = 2), S.hominis (n = 2), S.haemolyticus (n = 3) and S.
aureus (n = 1). The novobiocin resistance in S.saprophyticus strains
was not included in the evaluation as this species is inherently
resistant to this drug and no other isolates of Staphyloccocal
species were found to be resistant [29].
A possible example of the transfer of drug resistance
determinants occurring between different staphylococcal species
in the same restroom is shown with 7 isolates in restroom 16 K. All
7 isolates were antibiotic resistant at some level, but 6 of the
isolates, S. haemolyticus (n = 3), S. epidermidis (n = 2) and one S. aureus,
which were isolated from different sites within the same restroom,
had the same antibiogram. The two coagulase negative staphylococci, S. haemolyticus and S. haemolyticus also happen to be the two
of the most common CoNS found on man [30]. It is possible that
the 3 species with the same antibiogram came from the same
human source, as individuals undergoing long-term treatment for
acne can carry the more than one drug resistant staphylococcal
species [31]. These strains could then have been transferred to
different sites within the same restroom through poor hygiene
practices. However, this particular antibiogram was also found in
other Staphylococcal species isolated from 4 other restrooms
within the same building on different days, potentially indicating
a widespread dissemination of these resistance determinants
through different staphylococcal species and restrooms.
Other examples of similarities of antibiotic resistance determinants within these environments were found. Isolates of S.saprophyticus, for example were found with the same antibiograms in
different restrooms in the same building. Three S.hominis strains
were isolated from the same restroom and they also had the same
antibiogram and 2 S. pasteuri with the same antibiogram were also
isolated from different sites from different restrooms in the same
building. Although it is possible that contaminated individuals
were spreading these drug resistance determinants throughout
buildings, there is also the concern that antibiotic resistance is so
common in these environments and that it is easily spread. It

Table 4. Profiles of resistant Gram-negative isolates found in

restrooms.

Gram-negative isolates wr/B

Resistance Profiles

Sphingobacterium mizutaii 1 K

Am, Co, KF, S, ST, TS

D.acidovorans

11 K

A, Co, G, GM, N,S, ST, T, Tb

P.mirabilis

12 K

Co, KF, ST, TS

E. coli

18 K

Am, Am/S, GM, KF, S, ST, T,TS

A.baumannii

18 K

Am, C, Ct, Cf, KF, S, ST, T,TS

wr/B* restroom/building code G,K,H or T.

b - A: Amikacin; Am: Ampicillin; Am/S: Ampicillin/Sulbactam; KF: Cephalotoxin;
S: Streptomycin; ST: Sulphatriad; T: Tetracycline; TS: Cotrimoxazole; Co: Collstin
Sulphate C: Cefepime; Ct: Cefotaxime; Cf: Ceftazidime; G: Gentamicin; Tb:
tobramycin; N: Netilmicin.
doi:10.1371/journal.pone.0054223.t004

PBP29 Detection and PCR Amplification

In spite of the low MICs to methicillin in many staphylococcal
species, (mentioned above) we identified mecA in all of isolates
tested (Table 4).

Discussion
Our knowledge as too the variety of bacterial species which can
exist in microbiomes [7] is still developing and our understanding
about the spread and dissemination of antibiotic resistance within
such environments remains a challenge [8].
Bacteria shed from human skin, (Propionibacteriaceae, Corynebacteriaceae, Staphylococcaceae and Streptococcaceae) are common in restroom
environments [1] and these are not only the coliforms normally
targeted in hygiene screens [2,4,5]. In our study we isolated a wide
range of bacterial species from public restrooms and found
Staphylococcaceae were common in our study (Table 1) as they were
in the study by Flores et al [1]. Because of this we selected nonhealthcare restroom isolates of staphylococci to evaluate for their
propensity of drug resistance.
Staphylococci were isolated from 18 restrooms often on
different days. Previous studies have investigated non-healthcare
restrooms and homes but these targeted enteric pathogens and did
not evaluate levels of antibiotic resistance [2,4,5].
We propose that restrooms, especially with their continual
influx of bacterial flora from man, could be non-healthcare
environments for the collection of bacterial resistomes, which as
defined by Wright are a collection of antibiotic resistance genes in
pathogenic and non-pathogenic bacteria. Over a third of
staphylococci isolated in our study carried antibiotic resistance
determinants (Table 5).
Recently the bacterial phyla present in 12 selected restrooms
were comprehensively determined in a study by Flores and coworkers [1]. They demonstrated that organisms potentially shed
from human skin were most common in these environments in
particular, Propionibacteriaceae, Corynebacteriaceae, Staphylococcaceae and
Streptococcaceae and not the coliforms normally targeted in restroom
hygiene screens [2,4,5].
It was important to identify staphylococci to species level for our
study. For this we evaluated a number of methods. MALDI-TOFMS produced an identification rate similar to that found with
previous hospital studies (around 82 to 99%) [18,20,22]. PCR
agreed with MALDI-TOF-MS (99%). However the API ID 32
STAPH only produced only 54% agreement with the other
methods (Table 3). These figures were lower than those found
using API systems in clinical studies, but many of the species we
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Drug Resistant Bacteria in Nonhealthcare Restrooms

Table 5. Resistance profiles and molecular characterisation of antibiotic resistant Staphylococci isolated from 15 out of 18
different restrooms from 4 buildings (G,H,K,T).

Species

Wr/
B

A A
C C C
F
n A m z C l p x E F a G I

S. aureus

1K

1 R R R R R R R R

S. pasteuri

2T

S.epidermidis

4T

S.haemolyticus

4T

R R R R

R R

S. pasteuri

4T

R R R R R

S. hominis

4T

1 R R R R R

S.haemolyticus

5T

R R R R

R R

S. hominis

6K

R R

S. aureus

6K

1 R R R R R R R R

R R

S. simulans

7G

S.saprophyticus

7G

S. warneri

7G

2 R R R R R

S. hominis

9H

S. equorum

11 K

S. haemolyticus

11 K

S. warneri

11 K

S. hominis

11 K

S. warneri

M M M
S
T
L c p x N P S t T b O MIC

R R R

R R

R R R R
R R

R 2
0.25

64

R R R R

0.75

1.5

R R
R R R

R 2

R
R

R R
R R F R R

R R

R R R R

R R

Mec
A

R R R
R

R
R R

0.5

0.751.5

64

R R

0.75

R R

0.75

R R

R R

12 K

R R

0.75

S warneri

13H

128

S.saprophyticus

13H

R R R

1.5

S. pasteuri

14T

R R

R R

0.5

S. pasteuri

15 K

S.saprophyticus

15 K

R R

S. cohnii

16 K

S. aureus

16 K

S. epidermidis

16 K

S. haemolyticus

16 K

S. aureus

R R R

R R

R R

R R

R R

R R

0.51.5

R R

R R

0.51.5

17 K

R R

R R

64

S. epidermidis

18 K

128

S. capitis

18 K

R R

S.saprophyticus

18 K

R R

R R R

n* similar isolates from each restroom but different sites.

wr/B* restroom/Building code G,K,H or T.
A: Amoxacillin; Am: Ampicillin; Az: Azitromycin; C: Cefepime; Cx: Cefuroxime:; Cp: ciprofloxacin; Cl: Clindamycin; E: Erythromycin; F: Fosfomycin; Fa: Fusidic Acid; G:
Gentamicin; I: Imipenem; L: levofloxacin; Mp: Meropenem; Mx: Moxifloxacin; Mc: Mupiricin; N: Novobiocin; O: oxacillin; P: Penicillin; S: Streptomycin; T: Tetracycline. Tb:
tobramycin.
doi:10.1371/journal.pone.0054223.t005

carriage of mecA. By contrast, the five of the CoNS isolates,

although not multidrug resistant still carried mecA and demonstrated high MIC values (64 and 128mg/l) to oxacillin. In this
group with high oxacillin MICs were three strains of S.epidermidis,
one S.warneri and one S.equorum. These strains were only resistant to
oxacillin and penicillin. By comparison the only S.aureus strain in
this high resistance group was multiply drug resistant. This level
of oxacillin resistance has been also reported in community
acquired MRSA [35]. CAMRSA is well established throughout
the world, carriage and infections are often associated with close
communities such as students, military personnel and athletes [36].
Kummerer in 2004 [14] suggested that for the transfer of
resistance to occur, bacteria should be able to survive in the
environment and carry stable genetic material. There is also the

should also be noted that Coagulase-negative staphylococci can be

a significant problem in healthcare situations and in some
countries, have been reported to be the third most common
causative agent of nosocomial infections and the most frequent
cause of nosocomial bloodstream infections [32-34].
Our results showed that more than one third (37%) of
staphylococcal species isolated from our restroom samples were
carrying the mecA gene, although some had low MIC values to
oxacillin. Low MICs to oxacillin have also been reported in
clinical isolates of S.aureus [35] and S. haemolyticus [28] and these
isolates have also been reported as mecA positive.
Our findings, in common with those mentioned above,
demonstrate a commonality between low levels of oxacillin
resistance in clinical and/or in environmental isolates and the
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January 2013 | Volume 8 | Issue 1 | e54223

Drug Resistant Bacteria in Nonhealthcare Restrooms

proposal that antibiotic resistant bacteria can survive and persist

even in harsh environments [9]. Our study shows that bacteria,
not commonly associated with healthcare settings, carry resistance
determinants. Non-healthcare restrooms are a source of antibiotic
resistant bacteria. This shows the potential for public restroom
resistomes to exist, where a collection of antibiotic resistance
genes in pathogenic and non-pathogenic bacteria produce, as
suggested by Wright, a nexus of genetic diversity [37].

Author Contributions
Conceived and designed the experiments: RRC HM. Performed the
experiments: RRC HM CAR NW. Analyzed the data: RRC HM NW.
Contributed reagents/materials/analysis tools: RRC HM CAR NW.
Wrote the paper: RRC HM.

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